Anthraquinone inhibition of methane production in a ruminant animal

ABSTRACT

Anthraquinone compounds inhibit methane production by methanogenic bacteria in the rumen of ruminant animals, increasing production of volatile fatty acids and feed utilization efficiency. Preferred anthraquinones are unsubstituted anthraquinone, 1-aminoanthraquinone, 1-chloroanthraquinone, 2-chloroanthraquinone, 2-chloro-3-carboxyanthraquinone, 1-hydroxyanthraquinone, and 9,10-dihydroanthraquinone.

FIELD OF INVENTION

The invention relates to the use of anthraquinones as inhibitors ofmethane production in methanogenic bacteria.

BACKGROUND OF THE INVENTION

Regulation of methane production by methanogenic bacteria has severalimportant agronomic and environmental utilities. It has long beenrecognized that the regulation of methane production in cattle rumen hasaffected the efficiency with which cattle produce milk and beef fromfeedstocks. Additionally, there has been renewed environmental interestin the regulation of methane as a major greenhouse gas.

Microbial methane formation is a strictly anaerobic process which iscarried out by a metabolically unique group of organisms generally knownas the methanogenic bacteria. The group comprises the generaMethanococcus, Methanobacterium, Methanosarcina, Methanobrevibacter,Methanothermus, Methanothrix, Methanospirillum, Methanomicrobium,Methanococcoides, Methanogenium, and Methanoplanus. These bacteria arewidely distributed in strictly anaerobic habitats including the rumen ofruminant animals, the termite gut, landfills, stagnant ponds, anaerobicdigestors and rice paddies. The temperature range for growth may rangefrom mesophilic temperatures up to extremely thermophilic temperatures.

The methanogens are highly interactive ecologically, and depend heavilyon the metabolism of other bacteria to produce the substrates needed fortheir survival. Fermentative bacteria provide these substrates byconversion of complex macromolecules such as cellulose or protein intofour principal methanogenic substrates: hydrogen, carbon dioxide, aceticacid, and formic acid. The methanogens then remove these fermentativeend-products and convert them into gaseous methane and carbon dioxide.

The classic example of this type of association is termed "interspecieshydrogen transfer" wherein a hydrogen-producing organism generateshydrogen for the methanogen, and the methanogen then removes hydrogenwhich is actually inhibitory for the hydrogen producer. This is seen inthe natural food chain where primary bacteria convert cellulose tovarious products including lactate, acetate, fatty acids, carbon dioxideand hydrogen, and the methanogens then utilize the hydrogen and carbondioxide to produce methane and water.

In marine or brackish waters where sulfate is abundant, cellulose isconverted to carbon dioxide and hydrogen sulfide by sulfate reducingbacteria (SRB). These bacteria have a parallel metabolism to themethanogens and are able to utilize hydrogen and sulfate to producehydrogen sulfide. In sewage treatment facilities and in freshwater bogswhere sulfate concentrations are low, the SRB enter into a symbioticrelationship with the methanogens wherein the SRB produce hydrogen fromorganic acids and alcohols. The methanogens in turn convert the hydrogento methane and carbon dioxide.

Even though methanogens are typically grown in the laboratory under an80%/20% (vol/vol) hydrogen/carbon dioxide, in natural environmentsmethanogens and SRB are exposed to and grow on only traces of hydrogenand carbon dioxide. The intermediary levels of hydrogen, carbon dioxideand acetate may be very low but the methanogens and sulfate-reducers areable to grow on these substrates liberated by the fermentation ofsugars, organic acids (i.e., lactate, fatty acids) and alcohols.

There are at least two important utilities for inhibitors ofmethanogenesis. The first is the chemical manipulation of rumenfermentation as it occurs in ruminant animals such as cows and sheep, todivert microbial rumen metabolism away from methane formation and towardvolatile fatty acid formation. Methane represents a caloric loss to theruminant of 5-10% of its total caloric intake, and diversion of thisenergy into volatile fatty acids which the ruminant would use fornutrition would increase the efficiency of conversion of feedstocks intobeef. An inverse relationship between methane formation and productionof the volatile fatty acid, propionate, has been demonstrated by manyinvestigators, and therefore a positive effect of a methane inhibitor onrumen nutrition is expected. (C. J. Van Nevel, D. I. Demeyer,Manipulation of rumen fermentation. In: The Rumen Microbial Ecosystem,P. N. Hobson. (ed) Elsevier Publishing Co. (1988).)

Another important application of the inhibition of methane formationwould be a decrease in production of a major greenhouse gas andatmospheric pollutant.

Although methane constitutes only 0.4% of all greenhouse pollutants, itcontributes 18% of the total greenhouse warming of the earth'satmosphere, and its annual rate of increase is on the order of 1%. Someof the primary sources of environmental methane come from domesticanimals, landfills, and rice cultivation; which together contribute over40% of the total methane emissions and over 60% of the anthropogenicmethane emissions. Methane emissions from rice cultivation are estimatedto contribute about 20% of the total methane produced in the atmosphere,and emissions form landfills constitute about 7% of the total emissions.With respect to animal methane production, cattle are the ruminantsprimarily responsible for the largest methane emissions. The averagedairy cow may produce 200 liters of methane per day. The U.S. herd aloneproduces over 5 million metric tons of methane per year. Thus, theagricultural and industrial activities of man have become a significantcontributor to the total methane emission into the earth's atmosphere.

Methanogen inhibitors have been developed previously, primarily for useas feedstock additives to increase ruminant efficiency. Such additivesfall primarily into two classes. The first group are compounds whichindirectly affect methane formation by interfering with carbon orelectron flow at a point upstream of the methanogen in the microbialfood chain. The second group affects methanogens directly. Examples ofcompounds known to inhibit methanogenesis directly or indirectly arediverse, and range from common anions such as nitrate, to ionoporeantibiotics. Specific examples include monesin, lasalocid, salinomycin,avoparcin, aridcin, actaplanin, penicillin, chlorine and bromine methaneanalogs, long chain fatty acids, sulfate and nitrate. A complete list iscited in C. J. Van Nevel, D. I. Demeyer, Manipulation of RumenFermentation, In: The Rumen Microbial Ecosystem, P. N. Hobson (ed)Elsevier Publishing Co. (1988) hereby incorporated by reference. Clearlymost, if not all, of these compounds lack specificity for methaneformation, and some exhibit a multitude of side effects in the rumen ofanimals.

Numerous patents have been granted on a variety of compounds claiming todirectly or indirectly inhibit methane formation in ruminant animals. Itis believed that none, however, disclose use of anthracquinones asinhibitors of methane production.

The biological activities of anthraquinones are multitudinous and theutility of these compounds includes, for example, use as antimicrobials,proteolytic enzyme inhibitors, and as laxatives. The antimicrobialactivity of anthraquinone plant extracts such as Cassia sp. has beenlong recognized. The active component of Cassia has been identified as4,5 dihydroxyanthraquinone-2-carboxylic acid (Anchel, J. Biol. Chem.,177:169-177 (1949)). The existing literature, however, indicates thatthe general antimicrobial effects of anthraquinones appear to besporadic and unpredictable with regard to the bacterial species andprocesses affected. Studies have shown, for example, that some grampositive bacterial species such as Bacillus or Staphylococcus aresensitive to anthraquinone, but that gram negative bacteria such asEscherichia sp. or Pseudomonas sp. are insensitive (Kavanaugh, J.Bacteriol., 54:761-767 (1947)). However, other studies have shown thatthe 1,4,6,8 tetrahydroxyanthraquinone does not inhibit all strains ofBacillus, and that in Nocardia (gram positive) only one strain out offour is effected. The compound has no demonstrated effect on Escherichiacoli, Pseudomonas sp., Salmonella sp. or Sarcina sp. (Anke et al., Arch.Microbiol., 126:223-230 (1980); Anke et al., Arch. Microbiol.,126:231-236 (1980)). Metal Chelates of the 1,8 dihydroxyanthraquinonewere shown to be active against Bacillus subtilis, Bacillusstearothermophilus and Staphylococcus aureus whereas the 1,2dihydroxyanthraquinone and the 1-amino-4-hydroxyanthraquinone weregenerally inactive against these strains. The anthraquinones aloe-emodinand Rhein were found to be inhibitory to Bacillus subtilis andStaphylococcus aureus. However, the related anthraquinone, Chrysophanol,was not inhibitory to these strains. None of the anthraquinones testedinhibited the yeast Candida (Fuzellier et al., Ann. Pharm. Fr., 39(4)313-318 (1981)). Diaminoanthraquinones were shown to exhibit toxicityagainst gram positive cocci but not gram negative bacteria (Haran etal., Isr. J. Med. Sci., 17(6): 485-496 (1981)). These results typify thesporadic and unpredicatable antimicrobial effects of the anthraquinones.

Swiss Patent No. 614,466 discloses that anthraquinones with substituentmethyl, hydroxymethyl, carboxyl, aldehyde or carboxyethyl groups areknown to inhibit bacterial growth in tissue culture and in otherapplications where eukaryotic growth is desirable, but bacterial growthis not.

The 1,3,6,8 tetrahydroxyanthraquinone has been claimed as producing alaxative effect by stimulation of the neuromuscular junction of thebowel wall (U.S. Pat. No. 5,039,707).

Anthraquinones have also been shown to interfere with bacterial DNAmetabolism (Anke et al., Arch. Microbiol., 126:231-236 (1980)); and toinhibit ADP transport into mitochondria (Boos et al., FEBS Lett.,127:40-44 (1981)). The chemical reaction of reduced anthraquinone withoxygen to produce toxic superoxide radical may also be an importanttoxicity mechanism (Shcherbanoviskii et al., Rastit. Resur., 11(3):445-454 (1975)).

Miscellaneous inhibitory effects on particular enzyme systems have beenreported, but the overall lack of toxicity of anthraquinones issupported by their natural occurrence in plants, their widespread use asvat dyes for clothing and their use until recently as laxatives.Pharmaceutical use of anthraquinones, particularly hydroxylatedanthraquinones, has been curtailed due to the finding that they are weakmutagens. Halogenated anthraquinones, however, are not mutagenic (Brownet al., Mutation Research, 40:203-224 (1976)).

U.S. patent application Ser. No. 07/510,763, Pct publication No.91/15954 discloses that a large number of anthraquinone derivativesinhibit respiratory sulfate-reduction from anaerobic sulfate-reducingbacteria. Further, it was shown that other growth modes within thesebacteria were unaffected and that other bacterial types such asEscherichia coli and Saccharomyces sp. were unaffected by the preferredcompounds. The preferred anthraquinones comprised halogenated as well ashydroxylated derivatives. These compounds were shown to inhibit sulfideproduction in all laboratory strains of sulfate-reducing bacteria, aswell as crude sulfate-reducing enrichments from a variety of naturalenvironments.

In summary, although it has been shown that anthraquinones possess avariety of rather specific biological properties, these compounds havenever before been implicated as inhibitors of the methanogenic process.Use of these compounds fills a need, therefore, as inhibitors of methaneproduction from methanogenic bacteria. Preferably, this inhibitionshould be generally non-toxic, and have the ability to inhibit methaneproduction without significantly disrupting the natural equilibrium ofthe existing microbial population.

SUMMARY OF THE INVENTION

This invention provides a method of inhibiting methane production inmethogenic bacteria comprising contacting a medium containingmethanogenic bacteria with an anthraquinone compound. The methanogenicbacterial medium may be a mixed bacterial culture including, forexample, other hydrogen-producing or acetate-producing bacterialstrains. Preferably, the level of hydrogen present over the bacterialmedium is less than about 5% by volume, and the anthraquinone compoundsare present in the medium at a concentration of up to about 1 mg/liter.

The method of inhibiting methane production in methanogenic bacteria byaddition of anthraquinone, is useful, for example, to decrease themethane production in subsurface environments such as landfills, in ricepaddies, and in the rumen of ruminant animals. Such inhibition providesa method to reduce levels of a major greenhouse gas. This invention alsoprovides a method to increase production of volatile fatty acids inruminant animals, comprising feeding the ruminant animal ananthraquinone compound.

DETAILED DESCRIPTION OF THE INVENTION

The following terminology has been used by Applicant throughout thistext, and is offered for use in claim interpretation.

The term "anthraquinone compound" is defined to include anthraquinonecompounds comprising the basic tricyclic structure shown below, andincluding anthraquinone compounds substituted with up to four simplehalogen, carboxyl, hydroxyl, or amino substituents. We do not includetetracyclines or sulfonated anthraquinones exemplified by the reactivedyes. ##STR1##

Typical compounds included within the scope of the invention, forexample, include 1,8 diOH anthraquinone; 1 amino anthraquinone; 1 chloroanthraquinone; 2 chloro anthraquinone; 2 chloro-3carboxy anthraquinone;1 hydroxy anthraquinone; and anthraquinone.

The term "methanogenic bacteria" refers to bacteria which have theability to produce methane; including, but not limited to the generaMethanococcus, Methanobacterium, Methanosarcina, Methanobrevibacter,Methanothermus, Methanothrix, Methanospirillum, Methanomicrobium,Methanococcoides, Methanogenium.

The term "anaerobic digestor" refers to an apparatus used, for example,for the anaerobic conversion of municipal waste to methane and carbondioxide. See, Renand, P., Dochain, D., Bostin, G., Naveau, H., Nyns,B-J., "Adaptive Control of Anaerobic Digestion Processes: A Pilot ScaleApplication", Biotechnol. Bioeng., 31:287:294 (1988) which is hereinincorporated by reference.

The term "anaerobic digestor material" refers to material obtained, forexample, from municipal waste treatment facilities such as that usedherein, which is located in Wilmington, Del., and consisting ofmetabolizable organics, as well as microorganism such as fermentativeclostridia, methanogenic bacteria, and sulfate-reducing bacteria. See,for example, "Anaerobic Treatment Technology for Municipal andIndustrial Wastewater", M. S. Switzenbaum (Ed.), In: Water Science andTechnology, Vol. 24, No. 8 1991).

The term "methanogenic bacteria medium" as used herein refers to anymedium which permits growth of methanogenic bacteria. Specificallyincluded are defined laboratory cultures, and also any other manmade ornaturally occurring medium which permits methanogenic bacterial growthsuch as the anaerobic digestor material found in municipal wastetreatment digestors; landfills; rice paddies; the rumen of ruminantanimals; stagnant fresh water and marine ponds; or other naturallyoccurring or manmade anaerobic habitats.

A "ruminant animal" is one which derives it nutrition from theconversion of cellulose to volatile fatty acids. This occurs in aspecialized area of the digestive system referred to as the rumen. See,for example, C. J. Van Nevel, D. I. Demeyer, "Manipulation of RumenFermentation", The Rumen Microbial Ecosystem, P. N. Hobson. (ed)Elsevier Publishing Co. (1988).

One embodiment of the invention involves contacting a methanogenicbacteria medium with anthraquinone compounds, either in single or inmixed culture, under conditions wherein a steady state level of hydrogenis present at less than 5% by volume. Under these conditions it is seenthat levels of methane production are significantly reduced whencompared to cultures without anthraquinones. Construction of a definedtwo-membered bacterial population comprising laboratory strains of themethanogen and a hydrogen-producing organism will also produce thedesired conditions. A methanogenic, mixed culture enrichment obtainedfrom a naturally-occurring source such as an anaerobic digestor willalso create the desired conditions. The mixed methanogenic enrichmentfrom an anaerobic digestor, or rumen source, most closely approximatesthese ecosystems in terms of microbiological makeup.

Pure cultures of methanogens may be readily obtained from anyinternationally accredited biological repository such as the AmericanType Culture Collection, Rockville, Maryland, USA (ATCC). An example ofa pure strain of methanogen is M. formicicum corresponding to ATCCnumbers 33274.

Typical samples of anaerobic digestor material may be obtained fromwaste treatment plants which utilize anaerobic digestors. Methanogensare routinely cultivated in pure culture in the laboratory by growingthem in supportive medium under a gas phase comprising hydrogen/carbondioxide in an 80%/20% vol/vol ratio under argon or nitrogen with sodiumacetate. This growth condition does not approximate most conditionsoccurring in nature, but results in maximal growth rates and maximalcell densities. Samples of rapidly growing cultures maintained at thesehigh levels of hydrogen may then be transferred to fresh medium andtested for growth at various hydrogen concentrations. It has been foundthat cultures must be grown at low concentrations of hydrogencommensurate with those found in nature (less than 5%) for theanthraquinone to function as an effective methane inhibitor.

Concentrations of hydrogen and methane produced from single or mixedcultures are determined by standard methods known in the art, whereingas chromatography using Porapak® Q columns and argon carrier gas withthermal conductivity detection is preferred. Other suitable methods forhydrogen and methane measurement are described by Tadesse et al in J.Chromatogr., 171, 416, (1979) and Heidt et al., in J. Chromatogr., 69(1), 103, (1972).

The anthraquinone compounds of the instant invention are to bedistinguished from that broad group of anthraquinone-derivedantibiotics, typified by adriamycin, for example, which hasanthraquinone as a part of a much larger overall structure. Theanthraquinones comprising the instant invention include the basictricyclic structure show below, which may additionally be substitutedwith up to about four simple halogen, carboxyl, hydroxyl, or aminoderivative substituents. Typical examples of effective compounds areseen in Table 12. ##STR2## Generally, anthraquinones are not highlyreactive but undergo reversible oxidation-reduction.

Preferred anthraquinones in this application comprise9,10-dihyrdoanthraquinone, 1-amino anthraquinone, 1-chloroanthraquione,2-chloroanthraquinone, 2-chloro-3-carboxyanthraquinone,1-hydroxyanthaquinone and the unsubstituted anthraquinone; wherein theunsubstituted anthraquinone is most preferred. The anthraquinones foundto be effective inhibitors are all readily available commercialchemicals and do not require any special preparation other thandissolution in a suitable medium.

There are several accepted methods of preparing the anthraquinones fordelivery to the cultures. Finely divided particulate may be used fordelivering anthraquinones into the bacterial growth medium forinhibition of methanogenesis. Also, some anthraquinones may be dissolvedand added in liquid form. Water may be used to prepare aqueoussuspensions or solutions and these suspensions or solutions are mostpreferred for in vivo application of the invention. However, organicsolvents such as ethanol, methanol, dimethyl sulfoxide and acetone mayalso be used. These solvents will be the most preferred for experimentalconvenience in in vitro and laboratory applications. Of the organicsolvents, acetone is the most preferred. Once dissolved in theappropriate solvent the anthraquinones may be added directly to themethanogenic bacteria medium.

Effective final concentration of the anthraquinones is in the range of1-5 ppm (wt/vol=mg/1). It has been found that some anthraquinones aredegraded by the bacteria present in anaerobic digestors, and thus,repeated applications of the compound(s) may be necessary in order tomaintain the concentration of anthraquinone within the 1-5 ppm range.

The efficiency of feed utilization in domestic animals, especially theruminants such as cattle and sheep, is of economic importance in thefarming industry. It has been known for some time that compounds thatinhibit methanogenesis in ruminant animals also play a role in feedutilization.

As an aid to discovering methods of increasing the efficiency of feedutilization in ruminants, studies of the biochemical mechanisms by whichruminants digest and degrade food, particularly carbohydrates, has beenwidely studied. It is now known that carbohydrates are degraded in therumen to monosaccharides, which are converted to pyruvates, and then toacetates and propionates. Studies have shown that some of the rumenmicrobes ferment the monosaccharides of complex carbohydrates to formic,acetic, butyric and succinic acids, along with carbon dioxide andhydrogen. The carbon dioxide and hydrogen produced during fermentationare used in the formation of methane through the activity ofmethanogenic bacteria. These acetates, propionates and butyrates,collectively known as volatile fatty acids (or VTFA's), are all used asenergy sources by ruminants. However, the conversion of pyruvates toacetates involves chain-shortening by one carbon atom, and this carbonatom is lost in the form of carbon dioxide which is then irreversiblyconverted to methane gas. Since the production of propionic acid doesnot result in a loss of carbon but rather the incorporation of carbondioxide, the production of propionates from carbohydrates in the rumenof ruminant animals represents a more energy-efficient degradativepathway than the production of acetates and butyrates.

As a result, treatment of a ruminant so as to cause a shift in VTFAratios in the rumen towards increased rumen propionic acid leads to abeneficial effect on ruminant growth for a given amount of foodconsumption. Consequently, by improving the efficiency of rumenfermentation, a corresponding increase in the rate of growth and/or anincrease in the efficiency of feed utilization by the animals willoccur. (See U.S. Pat. No. 4,876,367.)

For instance, feed utilization efficiency and/or rate of growth canpreferably be improved by increasing the molar proportion of propionicacid to acetic acid or by increasing total volatile fatty acidconcentration (i.e., the sum of acetic, propionic and butyric acids) inthe rumen. In a similar fashion, it is also known that inhibitingmethanogenesis in the rumen results in an apparent decrease in gaseousloss of methane via eructation and a shift toward producing moredesirable fatty acids for growth, especially propionic and butyricacids. See U.S. Pat. Nos. 3,745,221; 3,615,649; and 3,862,333.

It is, therefore, a further object of the invention to provide compoundsand method for the inhibition of methanogenesis in ruminant animals withthe resulting beneficial effect of producing an increase in volatilefatty acids and an increase in feed utilization efficiency. Theanthraquinones of the present invention when contacted with crude rumenbacterial cultures were seen to decrease the levels of methane producedand to shift volatile fatty acids production in favor of proprionate.

In one preferred embodiment, runimal fluid is extracted from afistualted steer and a representative population of microorganisms isthereby obtained. Typically, a sample of rumen fluid is strained throughcheesecloth and the eluate is collected. The particulate matter retainedby the cheesecloth is resuspended in physiological buffer and the eluateis strained again. Buffers suitable for cell isolation are described byCheng et al., J. Dairy Sci. 38, 1225 (1955). Eluates are pooled andallowed to stand until particulate matter separates to the top. Theclear layer is then diluted with the same buffer, and adjusted to pH 7.0for use in incubations.

Methods for the determination of volatile fatty acids are well known inthe art. Typically, chromatographic methods such as HPLC or gaschromatography with flame ionization detection are preferred. Methodssuitable for use in the present invention are described by Jen et al.,in J. Chromatogr., 629(2), 394, (1993) and Nakamachi et al., in KogyoYosui, (391), 36, (1991).

As has been mentioned above, there are several compounds commerciallyavailable that are known to enhance production of desirable volatilefatty acids in ruminant animals, most notably monensin and 2,2,dichloroacetamide. (See U.S. Pat. No. 3,839,557.) To test the effectiveness ofanthraquinones on the production of desirable volatile fatty acids thesecompounds were used as positive controls in experiments where fatty acidproduction was analyzed.

The chemistry and microbiology of the rumen is complex and is affectedby many factors, not the least of which is the dietary intake of fiber.The production of desirable volatile fatty acids is highly dependent onthe presence of the appropriate ruminal microorganisms which are in turnaffected by the components of dietary intake. It has been seen forexample that ruminant microbial populations fluctuate broadly when sheepare fed diets of high-roughage containing corn meal and molasses, ascompared with alfalfa hay. Mackie, J. Agric. Sci., 103(1), 37, (1984).Allowing for the possibility that altered diets might impactmethanogenesis and VTFA production, fistulated steers were fed diets ofeither alfalfa hay or 50:50 forage concentrate diet consisting of 50%alfalfa and 50% ground corn. It was seen that methane production wasequally well inhibited under both dietary conditions, however, desirableVTFA production was only significantly increased in steers fed with the50:50 forage concentrate diet.

As is well known in the art liberation of ammonia nitrogen is a measureof proteolysis and, when applied to the contents of the rumen, anindirect measure of the rate of digestion. During the analysis of VTFAproduction the liberation of ammonia nitrogen was tracked using amodified colorimetric assay involving phenolhypochlorite and read at awavelength of 630 nm as described in Searcy et al., Clinica Chem. Acta.,12, 170, (1965).

In order to be effective feed additives in the field, active compoundsmust not only be able to enhance desirable volatile fatty acidproduction and inhibit methanogenesis but also be free of inhibitoryeffects on the rate of fiber digestion. To determine whether theanthraquinones of the present application had any effects that wouldinterfere with the digestive process, rates of digestion in thefistualted animals was determined by measuring the rate of digestion ofacid detergent fiber (ADF).

Routine methods of analysis of ADF are generally based on the ADF samplebeing pretreated such that the other components are solubilized bychemical degradation ("crude fibre", Official Methods of AOAC, 1975,136), or by treatment with wetting agents ("neutral detergent fiber/aciddetergent fiber", van Soest and Wine, J. AOAC, 1967, 50, 50), or byenzymatic degradation (Weinstock and Benham, J. Cereal Chem., 1951, 28,490; Hellendoorn et al., J. Sci, Food Agric., 1975, 26, 1461). Dietaryfibres are then separated by filtration of the sample through a glassfilter. Methods for determination of the rate of digestion using aciddetergent fiber are well known in the art. (See for example, Goering etal., Forage Fiber Analysis. Agriculture Handbook #3, (1970), AgricultureResearch Service, USDA, Washington, DC.)

The following nonlimiting examples are presented to illustrate severalof the important aspects of the present invention. Since methanogens arecommonly present at high levels in anaerobic digestor sludge and in therumen of cows, these two distinct ecosystems were chosen to demonstratethe effect of anthraquinones on methane production in these systems.Additionally, defined mixed cultures of bacteria, includingcharacterized methanogen strains, were investigated. The effect ofanthraquinones on other nonmethanogenic reactions, including glucosefermentation to hydrogen, and lactic acid fermentation to hydrogen, werecharacterized; as well as the methanogenic conversion of glucose orlactate to methane and conversion of hydrogen plus carbon dioxide oracetate to methane.

EXAMPLES Example 1 Methodology and Growth Conditions

A defined mineral medium of the following composition was used as abasal medium to which carbon and electron donors and electron acceptorswere added depending on the desired growth condition. This basal mediumis designated Medium BTZ-3 and is defined in Table 2.

                  TABLE 2                                                         ______________________________________                                        BTZ-3 Growth Medium                                                           Component           Concentration                                             ______________________________________                                        Ammonium chloride      4.3     g/l                                            Potassium dihydrogen phosphate                                                                       0.5     g/l                                            Magnesium chloride hexahydrate                                                                       0.20    g/l                                            Calcium chloride dihydrate                                                                           0.10    g/l                                            HEPES buffer (1.0 M)   50.0    ml                                             "Solution 1"           10.0    ml                                             0.2% Resazurin         1.0     ml                                             Deionized water        900     ml                                             ______________________________________                                    

The chemical components of "Solution 1" are given in Table 3. (HEPES isN-[2-hydroxyethyl]piperazine-N'-[2-ethonesulfonic acid]. Resazurin isused as a redox indicator and is not an obligatory part of the medium.)

                  TABLE 3                                                         ______________________________________                                        "Solution 1"                                                                  Component           Concentration                                             ______________________________________                                        Nitrilotriacetic acid  12.8    g/l                                            Ferrous chloride tetrahydrate                                                                        0.3     g/l                                            Cuprous chloride dihydrate                                                                           0.025   g/l                                            Manganous chloride tetrahydrate                                                                      0.1     g/l                                            Cobaltous chloride     0.32    g/l                                            Zinc chloride          0.1     g/l                                            Boric acid             0.01    g/l                                            Sodium molybdate       0.01    g/l                                            Nickel chloride        0.184   g/l                                            Deionized water        1000    ml                                             Adjust pH to 7.0 with 1M NaOH.                                                ______________________________________                                    

To prepare the BTZ-3 medium the components of Table 2 were mixed in around bottom flask and boiled under argon. The medium was then reducedby adding 40 ml of reducing agent to hot medium under argon. Thereducing agent consisted of 0.2N NaOH (1.6 g in 200 ml water) incombination with sodium sulfide nonhydrate (2.5 g/200 ml) and cysteinehydrochloride (2.5 g/200 ml). 20 drops of 1M HCL were added and the pHwas adjusted to pH 6.8-7.0. The medium was then dispensed into growthtubes or bottles, and argon gassing was continued in both growthcontainers and the medium. The medium was then sterilized by autoclavingfor 20 minutes at 115° C.

The reducing agent was prepared by boiling (0.2N) NaOH under argon,followed by cooling and adding sodium sulfide. After the sodium sulfidehad dissolved cysteine hydrochloride was added and permitted todissolve. The reducing agent was then dispensed under argon at 10 ml pertube and autoclaved for 20 minutes at 115° C.

Modifications to BTZ-3 were used, where noted, and usually consisted ofone or more of the following: sodium acetate, sodium lactate, yeastextract (Difco Laboratories), hydrogen/carbon dioxide gas phase. Allanthraquinones were added as 1000 ppm solutions in acetone with theexception of the 2-chloro, 3-carboxy anthraquinone which was added as a20 mM aqueous solution.

Example 2 Anaerobic Digestor Enrichment Studies

This example demonstrates the effect of the anthraquinones (AQ)1,8-dihydroxyanthraquinone, 9,10-dihydroanthraquinone, and2-chloroanthraquinone on fermentative and methanogenic stages of theanaerobic breakdown of lactate (column A) or glucose (column B) tomethane in anaerobic digestor sludge. This example investigated thefollowing stages of digestion:

1) The fermentation of glucose to hydrogen, acetate and carbon dioxide.

2) The fermentation of lactate to hydrogen, acetate and carbon dioxide.

3) The fermentation of glucose to hydrogen, acetate, carbon dioxide andmethane.

4) The fermentation of lactate to hydrogen, acetate, carbon dioxide andmethane.

Anaerobic digestor sludge was obtained from the Wilmington, DE wastetreatment facility and was enriched on glucose or lactate amended mediafor subsequent experiments. All media were amended with 0.05% yeastextract. After a preculture period of 24 hours, a 10% inoculum of thepreculture was transferred to the modified medium to start theexperiment. For this Example, the "Lactate, Column A" represents 30 mMsodium lactate in the BTZ-3 grown medium. The "Glucose, Column B"represents 10 mM glucose in the BTZ-3 growth medium.

1,8-dihydroxyanthraquinone, 9,10-dihydroanthraquinone, or2-chloroanthraquinone were then added to the cultures at the fourdifferent concentrations indicated in Table 4, and incubated for fourhours. The hydrogen and methane produced per hour in the culture weremeasured by gas chromatography using Porapak® Q columns and argoncarrier. Thermal conductivity detection was used. Results are given inTables 4-6.

                  TABLE 4                                                         ______________________________________                                        AO is 2-chloroanthraquinone                                                           umol H.sub.2 /h/culture                                                                   nmol methane/h/culture                                              A       B         A       B                                         AO in uM  Lactate Glucose   Lactate Glucose                                   ______________________________________                                        0         2.1     5.7       360     50                                        3.5       2.3     5.4       160     12                                        7         2.3     6.1        79     0                                         17.5      1.7     5.8       0.3     0                                         ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        AO is 1,8-dihydroxyanthraquinone                                                      umol H.sub.2 /h/culture                                                                   nmol methane/h/culture                                              A       B         A       B                                         AO in uM  Lactate Glucose   Lactate Glucose                                   ______________________________________                                        0         1.8     4.7       285     43                                        3.5       1.8     5.6       326     12                                        7         1.9     5.4       206     0                                         17.5      1.6     3.6       0.6     0                                         ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        AO is 9,10-dihydroxyanthraquinone                                                     umol H.sub.2 /h/culture                                                                   nmol methane/h/culture                                              A       B         A       B                                         AO in Mm  Lactate Glucose   Lactate Glucose                                   ______________________________________                                        0         1.45    6.2       320     --                                        3.5       2.1     6.2       295     39                                        7         2.1     5.9        97     43                                        17.5      2.2     4.2        26      0                                        ______________________________________                                    

This data demonstrates the general lack of effect of2-chloroanthraquinone, 1,8-dihydroxyanthaquinone, or the unsubstituted9,10-dihydroanthraquinone on fermentation of either lactate or glucoseto hydrogen. Methane formation from either lactate or glucose is,however, almost completely inhibited at 17.5 uM by all threeanthraquinones. This suggests that inhibition occurs at the point ofmethane formation from hydrogen and carbon dioxide or acetate (bymethanogenic bactiera), and not at the point of hydrogen or acetateformation (by fermentative organisms).

Example 3 Conditions for Methane Inhibition

The object of Example 3 was to examine the effect of2-chloroanthraquinone on methane formation from hydrogen and carbondioxide or acetate while varying the concentration of hydrogen over thecultures.

Methane formation was investigated in anaerobic digestor enrichments byfirst preculturing anaerobic digestor sludge in BTZ-3 mediumsupplemented with 10 mM sodium acetate under H₂ /CO₂ (80/20 vol/vol). Aportion of this preculture was then transferred to fresh medium, (whichhad not been supplemented with acetate H₂ /CO₂) to achieve inoculationof 10% vol/vol. 2-chloroanthraquinone ("AQ") was added to these testcultures at the concentrations indicated in Table 7, and the cultureswere subjected to a series of 0.5% to 80% hydrogen concentrations.Initial rates of methane formation from the culture were determined (gaschromatography, Porapak Q column, argon carrier, thermal conductivitydetection). The results are given below in Table 7.

                  TABLE 7                                                         ______________________________________                                        nmol methane/h/culture                                                        AQ in um                                                                              0.5% H.sub.2                                                                            2% H.sub.2                                                                             5% H.sub.2                                                                           40% H.sub.2                                                                          80% H.sub.2                          ______________________________________                                        0       518       518      518    442    700                                  3.5     64        33       20     596    885                                  7       24        22       40     565    910                                  17.5     0         0        2     565    965                                  ______________________________________                                    

The effect of 2-chloroanthraquinone on methanogenesis from acetate inanaerobic digestor enrichments was also investigated. The procedure wasperformed as above, except that the test cultures all contained 30 mMsodium acetate. 2-chloroanthraquinone was added to the cultures at thedifferent concentrations indicated in Table 8 and the production ofmethane was measured as above. The results are given below in Table 8.

                  TABLE 8                                                         ______________________________________                                        AO in uM    nmol methane/h/culture                                            ______________________________________                                        0           60                                                                3.5         79                                                                7           26                                                                14.5         0                                                                ______________________________________                                    

The above data indicate that 2-chloroanthraquinone inhibitsmethanogenesis from both hydrogen (Table 7) or acetate (Table 8)substrate. However, with hydrogen as a methanogenic substrate, methaneformation was inhibited only at low hydrogen concentrations (i.e., 0.5%,2%, 5%) and not at 40% or 80% hydrogen. This inhibition was apparent ateven the low concentration of AQ; 3.5 uM 2-chloroanthraquinone. Lowambient concentrations of hydrogen are typically found in anaerobicdigestors or the rumen of cattle (C. J. Van Nevel, D. I. Demeyer,Manipulation of rumen fermentation. In: The Rumen Microbial Ecosystem,P. N. Hobson. (ed) Elsevier Publishing Co. (1988) and Renand, P.,Dochain, D., Bostin, G., Naveau, H., Nyns, B-J., Adaptive control ofanaerobic digestion processes: a pilot scale application, Biotechnol.Bioeng., 31:287:294 (1988).)

The other key methanogenic substrate in nature is acetate. Using acetateas a substrate, the 2-chloroanthraquinone was also found to inhibitmethane formation as seen in Table 8.

Example 4 Rumen Methanogenesis Studies

Example 4 examined the effect of 2-chloroanthraquinone on methanogenesisby methanogenic bacteria using alfalfa as a carbon source, carried outby a rumen enrichment.

Fresh rumen fluid was obtained from a fistulated cow (a cow with asampling point surgically implanted into the rumen compartment, obtainedfrom University of Delaware, Dept. of Animal Science and AgriculturalBiochemistry) and maintained at approximately 40° C. until inoculationinto fresh medium in a ratio of 2:3 media:rumen fluid (vol:vol). Themedium consisted of the basal mineral medium BTZ-3 (of Example 1) plus0.38 g/l sodium chloride and 2.63 g/l sodium bicarbonate. 20 ml of thismixture was dispensed into 30 ml Wheaton bottles each containing 200 mgof finely divided alfalfa as the methanogenic substrate. The gas phasesupplied was nitrogen/carbon dioxide 80/20 (vol/vol). The cultures wereincubated at 40° C. with shaking. 2-chloroanthraquinone (AQ) was addedat four different concentrations as indicated in Table 9, and theconcentrations of methane, hydrogen, acetate and propionate weremeasured over time. Hydrogen and methane were monitored by intermittentsampling of the gas phase by gas chromatography (Porapak® Q column,argon carrier, thermal conductivity detection). Acetate and propionatewere monitored by liquid sampling and high pressure liquidchromatography on a Hamilton Polypore H column with 0.013M sulfuric acidas the mobile phase. The results for the 21 hour time point aretabulated below in Table 9.

                  TABLE 9                                                         ______________________________________                                        μMols of Total Product Formed After 21 Hours                               Product   Control 4 uM AO    8 uM AO                                                                              20 uM AO                                  ______________________________________                                        Hydrogen  0.37    0.77       1.5    2.7                                       Methane   237     275        228    225                                       Acetate   549     511        526    460                                       Propionate                                                                              120     119        120     96                                       ______________________________________                                    

The data demonstrated only a marginal effect of 2-chloroanthraquinoneover the range of 0-20 uM. An approximate 8-fold increase in hydrogen,and a small diminution of the acetate and propionate levels were seen.The increase in H₂ is a very sensitive indication of the onset ofmethane inhibition. Electrons are being diverted into H₂ productionrather than for CO₂ reduction to methane. The experiment was repeated at40 uM 2-chloroanthraquinone by subculturing the controls under thegrowth conditions described above. Again 200 mg of alfalfa per culturewas added and the cultures incubated at 40° C. for 24 hours. The datafor this experiment are shown below in Table 10.

                  TABLE 10                                                        ______________________________________                                        umols of Total Product Formed After 24 Hours                                  Product        Control 40 um (AO)                                             ______________________________________                                        Hydrogen        50     225                                                    Methane        174     2.5                                                    Acetate        166     215                                                    Propionate     200     151                                                    ______________________________________                                    

The data at 40 uM (10 ppm) 2-chloroanthraquinone clearly demonstratethat methane formation is inhibited, as expected, while the unutilizedhydrogen accumulates. There is a slight enhancement of acetate formationand some depression of propionate formation.

Example 5 Defined Mixed Culture Studies

Example 5 examined the effect of an equimolar 1-and2-chloroanthraquinone mix on methane production by a defined mixedculture consisting of a sulfate-reducing bacterium Desulfovibriodesulfuricans WADS (source is Wilmington, Del. anaerobic digestion,"WADS") and a methanogen Methanobacterium formicicum. In this exampleboth bacteria are present in a culture where lactate is serving as thecarbon source. Although it is known that anthraquinones will inhibitsulfate reducing bacteria when growing as a result of sulfate reduction,here Desulfovibrio desulfuricans WADS is not growing bysulfate-reduction but rather by fermentation of lactate to hydrogen, andthus the chloroanthraquinones have no effect on the organism. In thisculture, Methanobacterium formicicum is growing on hydrogen and carbondioxide produced by the D. desulfurican to produce methane.

Lactate undergoes the following transformation as a result of metabolismby Desulfovibrio desulfuricans:

2 lactate→2 acetate+4 hydrogen+1 carbon dioxide

and the resulting hydrogen is taken to methane and water byMethanobacterium formicicum according to the following scheme:

4 hydrogen+1 carbon dioxide→1 methane+2 water

The anthraquinone mix was added to these cultures as a solution inacetone, at the three different concentrations indicated in Table 11,and the levels of hydrogen and methane produced by the cultures weremeasured at daily intervals. The results appear in Table 11.

                  TABLE 11                                                        ______________________________________                                        Total uMol of CH.sub.4 or H                                                    ##STR3##                                                                     Control    AO at 0.05 ppm                                                                            AO at 0.1 ppm                                                                            AO at 0.2 ppm                               Days H.sub.2                                                                              CH.sub.4                                                                             H.sub.2                                                                             CH.sub.4                                                                            H.sub.2                                                                            CH.sub.4                                                                            H.sub.2                                                                            CH.sub.4                       ______________________________________                                        0    45      26    51     21   46   23    53   27                             1    25      65    67     20   71   22    23   25                             4    5      318    50     33   59   31    60   30                             6    0.4    410    36    137   48   69    47   45                             7    0.26   440    24    293   55   124   39   45                             8    0.34   382    15    561   45   253   40   46                             ______________________________________                                    

In the control cultures (no anthraquinones) hydrogen, generated by thesulfate-reducers, appears very rapidly at time zero and then disappearsas it is converted to methane by the methanogen. This rapid hydrogengeneration occurs in all cultures including the anthraquinone-treatedones. At 0.05 ppm AQ the conversion of hydrogen to methane by themethanogen is somewhat retarded, and hydrogen levels remain high in allcultures up to and including 0.2 ppm. It is also apparent that methaneproduction is increasingly inhibited with increasing anthraquinone; andat the 0.2 ppm AQ level production of methane is almost completelydiminshed.

Example 6 Effect of Different Anthraquinones

Example 6 investigated the effect of different anthraquinones onmethanogenesis from anaerobic digestor sludge wherein lactate wasprovided as the electron and carbon source.

Anaerobic digestor enrichments were prepared with BTZ-3 mediumcontaining sodium lactate at 30 mM essentially as described in Example2. Anthraquinones were added as solutions in acetone at a concentrationof about 20 mM and methane levels were measured as described previously,over time course indicated. The results are given in Table 12.

                  TABLE 12                                                        ______________________________________                                                  Total uMol Methane                                                  Anthraquinone                                                                             0 hr     16 hr  36 hr  56 hr                                                                              64 hr                                 ______________________________________                                        No AQ       14       13     27     46   39                                    1,8 Di OH-- 11       10     12     12   11                                    1 Amino-    11       15     13     14   14                                    1 Chloro-   12       --     16     20   20                                    2 Chloro-   11       14     14     11   17                                    2 Chloro- 3 Carboxy-                                                                      17       16     28     29   29                                    1 Hydroxy-   9       11     10     12   11                                    Unsubstituted                                                                              9        9     10      9   11                                    ______________________________________                                    

From the data it is evident that the addition of any anthraquinonederivative including the unsubstituted anthraquinone caused inhibitionof methane formation. The 2 chloro- 3 carboxy anthraquinone was theweakest inhibitor. The data possibly suggests that the basic tricyclicring structure is the component necessary for inhibition and thataddition of, for example, simple chloro-, hydroxy-, or amino-substituents does not either enhance or destroy this activity.

Example 7 Effects of Anthraquinone Compounds on Methane Production andVolatile Fatty Acid Levels from Ruminal Microorganisms Isolated fromSteers Fed on a 100% Forage Diet Alfalfa Hay

Isolation of Microorganisms:

Runminal microorganism were isolated essentially as described in Example4. Briefly, batch cultures of mixed ruminal microorganisms wereestablished from a fistulated steer fed a diet comprised of 100% alfalfahay (100% forage). The in vitro diet was ground through a 1 mm meshscreen and used at a rate of 0.375 g in 30 ml of culture fluid (15 ml ofruminal fluid and 15 ml of a standard ruminal buffer). Standard ruminalbuffer is well known in the art and suitable examples may be found inGoering et al., Forage Fiber Analysis. Agriculture Handbook #3, (1970),Agriculture Research Service, USDA, Washington, DC. Ruminal fluid wascollected 3 hours after feeding, strained through four layers ofcheesecloth and processed to recover the particulate-boundmicroorganisms under anaerobic conditions.

Preparation of Anthraquinones and Control Compounds:

The compounds tested included 9,10 anthraquinone, 2 chloroanthraquinoneand the 2 chloro-3 carboxy anthraquinone. Monensin (Sigma Chemical Co.,St. Louis, Mo.) and 2,2 dichloroacetamide (2,2 DCA) (Aldrich CehmicalCo., Milwaukee, Wis.) are two compounds currently used commercially asfeed additives for the purpose of methane inhibition and were used aspositive controls. All compounds were solubilized in ethanol, andappropriate dilutions prepared such that 0.25 ml of solution yieldedtargeted concentrations (ppm in the culture fluid). Control culturesreceived 0.25 ml of ethanol alone. Data (not shown) from previousstudies have shown minimal effects of this level of ethanol on rumenfermentation.

Incubation Conditions:

Incubations were performed anaerobically in 50 ml serum bottlesmaintained at 40° C. Three incubation replicates were prepared for eachcompound at each dose and incubations were typically for 24 hours.

Measurement of Gas and Volatile Fatty Acid Levels: After 24 hours ofincubation total gas production was measured by displacement and gassamples taken for future analyses. pH was determined immediately,followed by addition of 1 ml of 25% m-phosphoric acid to 5 ml offermentation fluid. The acidified fluid was analyzed for ammonianitrogen colorimetrically using a modified phenolhypochlorite method andread at 630 nm essentially as described in Searcy et al., Clinica Chem.Acta., 12, 170, (1965).

Volatile fatty acids were determined by gas chromatography (Model 589Hewlett-Packard Avondale, Pa.) using a 10 meter, 530 um macroboreCarbowax® M column (Supelco Inc. Bellefonte, Pa.). Helium at a flow rateof 10 ml/min was the carrier gas. One microliter of sample was injectedat 8:1 split ratio. Injection port temperature was 200° C. and detectortemperature was 250° C. Oven temperature program was 0° C. for 1 min, 5°C./min increase to 100° C., 45° C./min to 170° C. with a final holdingtime of 5 minutes. Volatile fatty acids (VTFA) measured included aceticacid (C2), propionic acid (C3), isobutyric acid (Ci4), isovaleric acid(C_(i) 5), and valeric acid (C5).

Methane and hydrogen were analyzed by intermittent sampling of the gasphase by gas chromatography (Porapak Q column, argon carrier, thermalconductivity detection). Initial oven temperature was 90° C. for 1minute followed by 30° C./min until a final temperature of 190° C. wasattained and held for 6 minutes. Argon was the carrier gas with a flowof 11 mV/min.

Effects of Anthraquinones on Gas and VTFA Production:

Table 13 shows the data collected from runminal microorganisms isolatedfrom a fistulated steer fed on a forage diet, treated with variousanthraquinone compounds, monesin and 2,2,dichloroacetamide.

                                      TABLE 13                                    __________________________________________________________________________    In vitro Effect of Anthraquinone Analogs, Monensin (M) and 2.2 Dicloro        Acetamide (2.2 DA)                                                            on Ruminal Fermentation Pattern of a 100% Forage Diet Alfalfa Hay             (Replicate 1)                                                                 Treatment                                                                             C.sub.2.sup.1                                                                    C.sub.3                                                                          C.sub.i.sup.4                                                                    C.sub.4                                                                          C.sub.i.sup.5                                                                    C.sub.5                                                                          TVFA.sup.2                                                                        Methane.sup.3                                                                      Hydrogen.sup.4                                                                     NH3 mg/dl                             __________________________________________________________________________    Time 0  40.02                                                                            8.80                                                                             0.65                                                                             3.51                                                                             0.96                                                                             0.75                                                                             54.69         11.46                                 Control 85.57                                                                            18.22                                                                            1.10                                                                             8.02                                                                             1.60                                                                             1.91                                                                             116.42                                                                            189.98                                                                             2.39 21.93                                 M   .5 ppm                                                                            74.90                                                                            19.74                                                                            1.17                                                                             7.02                                                                             1.15                                                                             1.92                                                                             106.27                                                                            116.53                                                                             1.8  22.48                                 2,2 DA                                                                            .5 ppm                                                                            79.91                                                                            17.57                                                                            1.05                                                                             8.11                                                                             1.51                                                                             1.84                                                                             109.99                                                                            111.51                                                                             1.56 21.89                                 AQ1.sup.5                                                                         .5 ppm                                                                            81.60                                                                            18.93                                                                            1.08                                                                             8.53                                                                             1.58                                                                             1.92                                                                             113.64                                                                            84.89                                                                              1.24 20.92                                     1 ppm                                                                             72.95                                                                            18.20                                                                            0.97                                                                             8.33                                                                             1.42                                                                             1.85                                                                             103.72                                                                            69.10                                                                              1.39 20.82                                     5 ppm                                                                             70.71                                                                            18.32                                                                            0.99                                                                             8.96                                                                             1.46                                                                             1.99                                                                             102.43                                                                            48.58                                                                              5.63 20.31                                 AQ2.sup.6                                                                         .5 ppm                                                                            73.70                                                                            17.82                                                                            0.94                                                                             7.96                                                                             1.37                                                                             1.79                                                                             103.58                                                                            92.59                                                                              1.53 20.70                                     1 ppm                                                                             69.92                                                                            20.82                                                                            0.85                                                                             8.68                                                                             1.27                                                                             1.96                                                                             103.50                                                                            31.72                                                                              7.6  22.19                                     5 ppm                                                                             72.55                                                                            19.66                                                                            1.04                                                                             8.35                                                                             1.56                                                                             1.95                                                                             105.11                                                                            45.01                                                                              10.04                                                                              21.31                                 AQ3.sup.7                                                                         .5 ppm                                                                            78.65                                                                            16.96                                                                            0.99                                                                             7.46                                                                             1.43                                                                             1.77                                                                             99.81                                                                             99.11                                                                              1.51 21.06                                     1 ppm                                                                             82.53                                                                            17.93                                                                            1.07                                                                             7.82                                                                             1.50                                                                             1.81                                                                             112.66                                                                            160.51                                                                             2.70 19.22                                     5 ppm                                                                             77.53                                                                            17.04                                                                            0.96                                                                             7.72                                                                             1.42                                                                             1.77                                                                             106.44                                                                            124.70                                                                             2.22 20.30                                 __________________________________________________________________________     .sup.1 mM of acetic (C2), propionic (C3), isobutyric (Ci.sup.4),              isovaleric (C.sub.i.sup.5), and valeric (5), acids.                           .sup.2 Total violatile fatty acids.                                           .sup.3 uMoles of methane produced.                                            .sup.4 uMoles of hydrogen produced.                                           .sup.5 2chloro anthraquinone.                                                 .sup.6 9,10 anthraquinone.                                                    .sup.7 3 chloro, 2 carboxy anthraquinine.                                

Table 13 shows that 9,10 anthraquinone, 2-chloroanthraquinone and the 2chloro-3 carboxy anthraquinone all inhibited methane formation with thecarboxylated derivative being least effective. Little effect was seen onhydrogen formation except at 5 ppm levels of the 9,10 anthraquinone.Acetate formation was suppressed by the 9,10 anthraquinone and 2chloroanthraquinone. Only a slight elevation in propionate and butyratelevels was seen. Monensin, a commercial methane inhibitor (andpropionate-enhancer) inhibited methane formation at 0.5 ppm but had onlya marginal effect on fatty acids at this concentration. The same isobserved for the rumen additive 2,2 dichloroacetamide. Total volatilefatty acid production appears to be very slightly suppressed in mosttreated incubations relative to untreated controls.

The data shown here demonstrate that with a forage diet (alfalfa hay)there is a definite inhibition of methane formation by 0.5 to 5 ppmlevels of the three anthraquinones tested with the unsubstituted and2-chloro- denvatives being the best. Methane inhibition by theanthraquinones appears to be at least as good if not better thanmonensin or 2,2 dichloroacetamide. None of the compounds tested impactedvolatile fatty acids significantly. There appears to be no discernibleadverse effect of anthraquinones or the other compounds tested onproteolysis as indicated by the levels of free ammonia nitrogendetermined in the incubation.

Example 8 Effects of Anthraquinone Compounds on Methane Production andVolatile Fatty Acid Levels from Ruminal Microorganism Isolated fromSteers Fed on a 50:50 Forage Concentrate Diet

Microorganisms were isolated exactly as described in Example 7 however,in this instance ruminal microorganisms were isolated from a fistulatedsteer fed a diet comprised 50:50 forage concentrate diet which wascomprised of 50% alfalfa and 50% ground corn. Preparation of compounds,incubations and gas and VTFA measurements were done essentially asdescribed in Example 7. Data demonstrating the effect of anthraquinonescompounds on gas and VTFA production is given in Table 14.

                                      TABLE 14                                    __________________________________________________________________________    In Vitro Effect of Anthraquinone Analogs, Monensin (M) and 2.2 Dicloro        Acetamide (2.2 DA) on Ruminal Fermentation Pattern of a                       50:50 Forage:Concentrate Diet (replicate 1)                                   Treatment                                                                             C.sub.2.sup.1                                                                    C.sub.3                                                                          C.sub.i.sup.4                                                                    C.sub.4                                                                          C.sub.i.sup.5                                                                    C.sub.5                                                                          TVFA.sup.2                                                                        Methane.sup.3                                                                      Hydrogen.sup.4                                                                     NH3 mg/dl                             __________________________________________________________________________    Time 0  34.5                                                                             12.10                                                                            0.77                                                                             8.98                                                                             1.69                                                                             1.14                                                                             59.18                                               Control 66.70                                                                            20.78                                                                            1.00                                                                             18.66                                                                            2.20                                                                             2.47                                                                             111.85                                                                            286.09                                                                             4.36                                       M   .5 ppm                                                                            63.73                                                                            35.69                                                                            0.82                                                                             15.69                                                                            2.06                                                                             2.46                                                                             120.45                                                                            150.69                                                                             7.25                                       2,2 DA                                                                            .5 ppm                                                                            65.83                                                                            30.11                                                                            1.08                                                                             20.79                                                                            2.26                                                                             2.75                                                                             122.82                                                                            152.31                                                                             6.15                                       AQ1.sup.5                                                                         .5 ppm                                                                            67.15                                                                            30.38                                                                            1.11                                                                             20.94                                                                            2.31                                                                             2.79                                                                             121.89                                                                            185.42                                                                             4.20                                           1 ppm                                                                             62.58                                                                            31.21                                                                            1.00                                                                             21.90                                                                            2.15                                                                             2.68                                                                             121.52                                                                            114.48                                                                             31.81                                          5 ppm                                                                             56.86                                                                            29.80                                                                            0.87                                                                             25.84                                                                            1.95                                                                             2.85                                                                             118.17                                                                            56.41                                                                              138.14                                     AQ2.sup.6                                                                         .5 ppm                                                                            69.15                                                                            31.05                                                                            1.11                                                                             21.80                                                                            2.31                                                                             2.79                                                                             128.21                                                                            177.75                                                                             4.12                                           1 ppm                                                                             61.70                                                                            32.23                                                                            0.93                                                                             22.94                                                                            2.04                                                                             2.74                                                                             122.58                                                                            72.75                                                                              91.82                                          5 ppm                                                                             55.96                                                                            30.54                                                                            0.85                                                                             25.53                                                                            1.91                                                                             2.80                                                                             117.59                                                                            13.33                                                                              217.29                                     AQ3.sup.7                                                                         .5 ppm                                                                            75.83                                                                            28.02                                                                            1.13                                                                             20.67                                                                            2.37                                                                             2.62                                                                             130.64                                                                            244.75                                                                             3.31                                           1 ppm                                                                             76.61                                                                            27.52                                                                            1.08                                                                             20.76                                                                            2.32                                                                             2.63                                                                             130.92                                                                            246.69                                                                             3.69                                           5 ppm                                                                             72.30                                                                            25.60                                                                            1.01                                                                             21.74                                                                            2.23                                                                             2.60                                                                             125.48                                                                            237.09                                                                             3.14                                       __________________________________________________________________________     .sup.1 mM of acetic (C2), propionic (C3), isobutyric (Ci.sup.4), butyric      (C4), isovaleric (C.sub.i 5), and valeric (5), acids.                         .sup.2 Total violatile fatty acids.                                           .sup.3 uMoles of methane produced.                                            .sup.4 uMoles of hydrogen produced.                                           .sup.5 2chloro anthraquinone.                                                 .sup.6 9,10 anthraquinone.                                                    .sup.7 3 chloro, 2 carboxy anthraquinine.                                

Table 14 shows the effect of anthraquinones, monensin and 2,2dichloroacetamide on fermentation of a 50:50 mixture of hay:feedlotconcentrate diet. This diet more closely approximates that to be used inactual application of rumen additives. Methane is clearly suppressedeven at 0.5 ppm levels of the compounds tested with the exception of thecarboxylated anthraquinone which showed only minimal impact on methaneformation. In contrast to the 100% alfalfa incubations illustrated inExample 7, large accumulations of hydrogen were observed in the 9,10anthraquinone or 2-chloro- treated incubations. Volatile fatty acids,particularly acetate, propionate and butyrate were clearly affected.Acetate formation was suppressed whereas propionate and butyrateformation were enhanced. These enhancements are favorable to themetabolism of the ruminant animals and may result in increasedefficiency of growth. Ammonia nitrogen was not determined in theseparticular experiments. Long term adaptation of the culture to theexcess production of hydrogen should, in theory result in enhancedpropionate and butyrate formation since formation of these compoundsrequires reductant often in the form of hydrogen gas.

Example 9 Effect of Anthraquinones on Fiber Digestion

It is important that the experimental compounds not interfere with fiberdigestion. Accordingly, 9,10 anthraquinone, 2 chloroanthraquinone, 2chloro-3 carboxy anthraquinone, monensin and 2,2 dichloroacetamide weretested for their effect on fiber digestion of a feed comprised of 100%alfalfa.

Fiber digestion was measured after 24 hours of incubation by analyzingthe feed residue for acid detergent fiber (ADF) content. Digestion ofADF was calculated by subtracting the residual ADF from the initialamount of ADF in the diet. Methods for calulcating Digestion of ADF arewell knonw in the art and examples may be found in Goering et al.,Forage Fiber Analysis. Agriculture Handbook #3, (1970), AgricultureResearch Service, USDA, Washington, DC.

As shown in Table 15, percent digestion is slightly lower in most of thetreated incubations. However there is no dose-response relationshipapparent with any anthraquinone. Therefore, we conclude that there is nosignificant effect of anthraquinone on fiber digestion. Controlsubstance was the solvent alone, used to dissolve the compounds ofinterest.

                  TABLE 15                                                        ______________________________________                                        Compound        Concentration                                                                            % Digestion                                        ______________________________________                                        Control         0          34%                                                Monensin        0.5 ppm    28%                                                2,2 DCA         0.5 ppm    35%                                                2-Chloro AQ     0.5 ppm    24%                                                2-Chloro AQ     1.0 ppm    26%                                                2-Chloro AQ     5.0 ppm    26%                                                9,10 AQ         0.5 ppm    32%                                                9,10 AQ         1.0 ppm    20%                                                9,10 AQ         5.0 ppm    26%                                                3-Chloro-2-Carboxy AQ                                                                         0.5 ppm    20%                                                3-Chloro-2-Carboxy AQ                                                                         1.0 ppm    22%                                                3-Chloro-2-Carboxy AQ                                                                         5.0 ppm    26%                                                ______________________________________                                    

What is claimed is:
 1. A method for increasing production of volatilefatty acids and inhibiting methane production in a ruminant animal, themethod comprising feeding the ruminant animal an anthraquinone compound.2. The method of claim 1 in which the ruminant animal is a cow.
 3. Themethod of claim 2 in which the anthraquinone compound is unsubstitutedanthraquinone.
 4. The method of claim 1 in which the anthraquinonecompound is selected from the group consisting of unsubstitutedanthraquinone, 1-aminoanthraquinone, 1-chloroanthraquinone,2-chloroanthraquinone, 2-chloro-3-carboxyanthraquinone,1-hydroxyanthraquinone, and 9,10-dihydroanthraquinone.
 5. The method ofclaim 4 in which the ruminant animal is a cow.
 6. A method forincreasing production of volatile fatty acids in a ruminant animal, saidanimal having a rumen and said rumen having a medium containingmethanogenic bacteria, the method comprising contacting the medium inthe rumen with an anthraquinone compound.
 7. The method of claim 6 inwhich the anthraquinone compound is selected from the group consistingof unsubstituted anthraquinone, 1-aminoanthraquinone,1-chloroanthraquinone, 2-chloroanthraquinone,2-chloro-3-carboxyanthraquinone, 1-hydroxyanthraquinone, and9,10-dihydroanthraquinone.
 8. The method of claim 7 wherein theanthraquinone compound is present in the medium at a concentration of upto 1 mg/L.
 9. The method of claim 7 in which the ruminant animal is acow.
 10. The method of claim 9 in which the anthraquinone compound isunsubstituted anthraquinone.
 11. The method of claim 6 in which theruminant animal is a cow.
 12. The method of claim 6 in which themethanogenic bacteria are selected from the group consisting ofMethanococcus, Methanobacterium, Methanosarcina, Methanobrevibacter,Methanotherms, Methanothrix, Methanospirllum, Methanomicrobium,Methanococcides, Methanogenium, and Methanoplanus.
 13. The method ofclaim 12 wherein the anthraquinone compound is selected from the groupconsisting of unsubstituted anthraquinone, 1-aminoanthraquinone,1-chloroanthraquinone, 2-chloroanthraquinone,2-chloro-3-carboxyanthraquinone, 1-hydroxyanthraquinone, and9,10-dihydroanthraquinone.
 14. The method of claim 6 in which a level ofhydrogen over the medium is less than 5% by volume.
 15. The method ofclaim 6 in which an amount of the anthraquinone compound in the mediumis sufficient to produce a concentration of up to 1 mg/L.